Our earlier studies have indicated that the endothelium-dependent vasodilator effect of the anandamide and of some atypical cannabinoid ligands, such as abnormal cannabidiol (abn-cbd) is mediated by a pertussis toxin-sensitive, G protein-coupled receptor distinct from CB1 or CB2. In collaboration with Mechoulam's group we have earlier identified a novel, endocannabinoid-like brain lipid, arachidonoyl L-serine (ARA-S). Contrary to anandamide, ARA-S has no affinity for CB1, CB2 or vanilloid TRPV1 receptors, and it produces partially endothelium-dependent vasodilation in rat isolated mesenteric artery and aorta preparations. In a more recent, ongoing study, we have analyzed the effect of ARA-S on Ca2+-activated K+ currents in human embryonic kidney cells stably transfected with the alpha-subunit of the human, large conductance Ca2+-activated K+ (BKCa) channel (HEK293hSlo cells). ARA-S caused relaxation of isolated, intact and denuded, small mesenteric arteries (pEC50: 5.49 and 5.14, respectively) of the rat. In both preparations the response was inhibited by 100 nM iberiotoxin. In HEK293hSlo cells, ARA-S and its enantiomer N-arachidonoyl-D-serine enhanced the whole cell outward K+ current with similar potency (pEC50: 5.63 and 5.32, respectively). The potentiation was not mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases or Ca2+-dependent processes, and it was lost after patch excision or following membrane cholesterol depletion, but was restored after cholesterol reconstitution. BKCa currents were also enhanced by anandamide (AEA, pEC50: 5.27) but inhibited by another endocannabinoid, virodhamine (pIC50: 6.35), or by the synthetic cannabinoid O-1918, which blocks ARA-S-induced vasodilation (pIC50: 6.59). These findings indicate that (i) endocannabinoids directly modulate the activity of BKCa channels or a channel-associated component. (ii) This interaction does not involve cytosolic factors but is dependent on the presence of membrane cholesterol (iii) Direct BKCa channel activation likely contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. (iv) Depending on the structure of the head group, the effect on BKCa currents is either stimulatory or inhibitory. (v) O-1918 is a potent BKCa channel inhibitor.[unreadable] [unreadable] We have earlier established that the vasodilated state in advanced liver cirrhosis is mediated by endocannabinoids acting at vascular CB1 receptors (Nat Med 7:827, 2001). Cirrhosis is also known to be associated with altered cardiac function characterized by reduced contractility and contractile response to catecholamines. The cirrhotic 'cardiomyopathy' was recently proposed to be also due to stimulation of CB1 receptors by endocannabinoids, based on a study using isolated papillary muscles from control and cirrhotic rats. In a recently published study, we have tested this hypothesis by examining the cardiovascular effects of CB1 antagonists in an in vivo model of CCl4-induced cirrhosis in rats, in which cardiac hemodynamics were analyzed directly by using the Millar pressure/volume system, and by post-mortem quantification of cardiac endocannabinoid and CB1 receptor content in cirrhotic rats and their controls. Rats with CCl4-induced cirrhosis developed decreased cardiac contractility, low blood pressure and tachycardia. Bolus i.v. injection of the CB1 antagonist AM251 (3 mg/kg) acutely increased mean blood pressure as well as both load-dependent and -independent indices of systolic function, whereas no such changes were elicited by AM251 in control rats. Furthermore, tissue levels of the endocannnabinoid anandamide increased 2.7-fold in the heart of cirrhotic compared to control rats without any change in 2-arachidonoylglycerol (2-AG) levels, whereas in the cirrhotic liver both 2-AG (6-fold) and anandamide (3.5-fold) were markedly increased. CB1 receptor expression in the heart was unaffected by cirrhosis, as verified by Western blotting. These results indicate that activation of cardiac CB1 receptors by endogenous anandamide contributes to the reduced cardiac contractility in liver cirrhosis, and CB1 receptor antagonists may be used to improve contractile function in cirrhotic cardiomyopathy and, possibly, in other forms of heart failure. [unreadable] [unreadable] We have also analyzed the potential involvement of the endocannabinoid system in another form of cardiomyopathy, the heart failure induced by treatment with doxorubicin. Doxorubicin is one of the most potent antitumor agents available; however, its clinical use is limited because of the risk of severe cardiotoxicity. Because of the known, CB1 receptor-mediated cardiodepressive effects of endocannabinoids and their contribution to myocardial dysfunction of various ethiologies, we examined their role in a mouse-model of doxorubicin-induced heart failure. Left ventricular function was measured by Millar pressure-volume system. Apoptosis markers, CB1/CB2 receptor expression, and endocannabinoid levels were determined by immunohistochemistry, Western blot, RT-PCR and real-time PCR, flow cytometry, fluorescent microscopy, and LC/MS techniques. Five days after the administration of a single dose of 20 mg/kg doxorubicin i.p., load-dependent and -independent indicators of left ventricular contractility as well as cardiac output were significantly depressed, and the myocardial level of the endocannabinoid anandamide (but not CB1/CB2 receptor expression) was elevated compared with vehicle-treated control mice. Treatment with the CB1 antagonists rimonabant or AM281 markedly improved cardiac dysfunction and reduced doxorubicin-induced apoptosis in the myocardium. Doxorubicin also decreased cell viability and induced apoptosis in the H9c2 myocardial cell line measured by flow cytometry and fluorescent microscopy, which were prevented by the preincubation of the cells with either CB1 antagonist, but not with CB1 and CB2 agonists or CB2 antagonists. These findings indicate that activation of cardiac CB1 receptors by anandamide contributes to doxorubicin-induced cardiotoxicity, and suggest that CB1 antagonists may represent a new cardioprotective strategy in this condition.[unreadable] [unreadable] Hepatic ischemia-reperfusion (I/R) is a fatal complication that can follow liver surgery or transplantation. We have investigated the involvement of the endocannabinoid system in hepatic I/R injury using an in vivo mouse model. We reported that I/R triggers several-fold increases in the hepatic levels of anandamide and 2-arachidonoylglycerol, which originate from hepatocytes, Kupffer, and endothelial cells. The I/R-induced increased tissue endocannabinoid levels positively correlate with the degree of hepatic damage and serum TNF-alpha, MIP-1alpha, and MIP-2 levels. Furthermore, a brief exposure of hepatocytes to various oxidants (H2O2 and peroxynitrite) or inflammatory stimuli (LPS, TNF-alpha) also increases endocannabinoid levels. Treatment with the CB2 agonist JWH133 protects against I/R damage by decreasing inflammatory cell infiltration, tissue and serum TNF-alpha, MIP-1alpha and MIP-2 levels, tissue lipid peroxidation, and expression of ICAM-1 in vivo. JWH133 also attenuates the TNF-alpha-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and the adhesion of human neutrophils to HLSECs in vitro. Consistent with the protective role of CB2 receptor activation, CB2-/- mice develop increased I/R-induced tissue damage and proinflammatory phenotype. Thus, targeting CB2 cannabinoid receptors may represent a novel protective strategy againust hepatic I/R injury.